Missense mutations within the multi-domain kinase, LRRK2, are the most common cause of familial Parkinson's disease (PD), accounting for up to 40% of cases in some populations. LRRK2 mutations also appear in an unprecedented number of sporadic PD patients, implicating this protein in all forms of PD. While the genetic links between LRRK2 dysfunction and PD pathogenesis are clear, less is known about the physiological activity of LRRK2 or how it is regulated. The weak penetrance of LRRK2 disease mutations, however, does suggest that other genes may potently influence LRRK2 function and play a role in its regulation. Recent data indicate that like other kinases, LRRK2 can form a functional dimer within the cell. We've shown that the LRRK2 dimer is several-fold more active than its monomeric counterpart. In addition, the majority (~75%) of total LRRK2 is found within the cytoplasm at rest, while the membrane fraction is selectively enriched in the LRRK2 dimer. This is highly reminiscent of other proteins involved in intracellular signaling, where various kinases and GTPases shuttle to and from the membrane, and can be activated through dimerization. Therefore, we hypothesize that these newly discovered biochemical properties of LRRK2 are consistent with canonical mechanisms used throughout nature to regulate kinase activity and intracellular signaling. However, the precise importance of these processes to LRRK2 are not known. Our long-term goal is to uncover the biochemical nature of PD-linked LRRK2 mutants. However, this has remained elusive, partly due to our incomplete understanding of LRRK2 biology. At this time, a broader understanding of the physiological function and regulation of wild-type LRRK2 is critically needed. The goal of this application is to optimize a newly developed, quantitative assay of LRRK2 dimerization, and use this tool to uncover critical aspects of LRRK2 regulation. Specifically, we will 1) develop a protein-fragment complementation assay of LRRK2 dimerization, and 2) identify cellular and genetic determinants of LRRK2 dimerization in the intact cell. This work will not only inform us as to the pathways involved in intracellular LRRK2 signaling, but also provide novel tools to examine the underlying mechanisms of several pathogenic LRRK2 mutations in future studies. PUBLIC HEALTH RELEVANCE: Mutations in the LRRK2 kinase are the most common genetic cause of Parkinson's disease. The goal of this application is to optimize a novel, high-throughput assay of LRRK2 biology to uncover how cells regulate LRRK2 function. Through this new resource, and an improved understanding of the pathways controlling LRRK2 activity, we hope to gain valuable insights into the physiological and pathological functions of LRRK2.